Minsun Jeon's CIAP Aptamer Project

Over the next two decades, the number of new cases of cancer is expected to rise by about 70% .1 As cancer develops from one cell, it can spread to all parts of the human body such as the joints and the organs through the process called metastasis. As shown in Fig. 2, cancer cells divide quickly, being unrestrained. If this process is not prevented fast enough, it can lead to a high risk of mortality. In attempts to subsidize cancer and to reduce the mortality rate, many scientists and researchers have been developing new drugs and advanced cancer treatments.

Although chemotherapy, a widely known cancer therapy that uses drugs to destroy cancer cells, is known to be effective, it also has side effects. Even though chemotherapy kills the cancer cells as desired, it also kills the healthy cells as well. Also, treatment of solid tumors with conventional cytotoxic drugs lack selectivity and thus results in toxic dose-limiting side effects.2 However, the concept of antibody-enzyme conjugates for cancer therapy specifically called “antibody-directed enzyme prodrug therapy”(ADEPT) can solve this problem and effectively administer cancer treatments.

The antibody-enzyme conjugates directed at tumor-associated antigens can be used to achieve site-specific activation of prodrugs to potent cytotoxic species.3 The protein target chosen for the aptamer selection, β-glucuronidase, may be a good candidate for conjugation to monoclonal antibodies (which recognizes a single, specific site of an agent of interest) to induce selective activation of prodrugs at the target site.4 A prodrug can be enzymatically converted to antineoplastic agent (which inhibits the growth of malignant cells) at tumors cells that are able to bind β-glucuronidase-monoclonal antibody conjugates. The targeted-β-Glucuronidase activation of a prodrug can increase the specificity of chemotherapy.5

Human β-Glucuronidase (GUS-his) is a member of the glycosidase family of enzymes that catalyzes the breakdown of complex carbohydrates through the hydrolysis of β-D-glucuronic acid residues from the non-reducing end of mucopolysaccharides. This enzyme is found in the human body, specifically located in the lysosome. It can also be found in various microorganisms such as E.coli, fungi, and protozoa. Functioning as a tetramer, as shown in Fig. 3, β-glucuronidase has a molecular weight of 57KDa. β-glucuronidase is negatively charged in its storage buffer of pH 7.4, Phosphate Buffered Saline Buffer, with a charge of approximately -21.2. Since it has a negative charge, it is less likely to bind to the DNA, which has a negative charge, due to the repulsion of the charges (It has an isoelectric point of 5.76).

In addition to the protein’s potential use in chemotherapy delivery, it can also be used to detect Mucopolysaccharidosis Type VII, which is a progressive condition that affects the tissues and organs.6 The main labs doing research on this target is currently working with plants to use the target to detect other targets of interests in the plant.

An aptamer is a nucleic acid binding species that has a binding affinity for a specific molecular target such as a protein or a small molecule. Through in vitro selection, aptamers are selected with the unbound DNA being filtered out, leaving the only bound DNA. An aptamer displays a tight binding and high specificity to cognate analytes and it is capable of discriminating between related targets on basis of a single amino acid or functional group. By binding to biomedically relevant proteins, aptamers exhibit significant advantages relative to protein therapeutics in terms of size, synthetic accessibility and modification by medicinal chemistry.7

There has not been an aptamer developed for the target, β-glucuronidase, but the future development of this aptamer can serve as a drug delivery application leading to more effective cancer therapy. A RNA aptamer selection against β-glucuronidase will be performed for the development of a possible aptamer to help site-specific activation of prodrugs through the antibody-enzyme conjugates.

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Citations

(1) WHO | Cancer. (n.d.). Retrieved April 10, 2015, from

http://www.who.int/mediacentre/factsheets/fs297/en/

(2) De Graaf, M., Boven, E., Oosterhoff, D., van der Meulen-Muileman, I. H.,

Huls, G. A., Gerritsen, W. R., … Pinedo, H. M. (2002). A fully human

anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective

chemotherapy with a glucuronide prodrug. British Journal of Cancer,

86(5), 811–818. http://doi.org/10.1038/sj.bjc.6600143

(3) Melton, R. G., & Sherwood, R. F. (1996). Antibody-Enzyme Conjugates for

Cancer Therapy. Journal of the National Cancer Institute, 88(3-4), 153–

165. http://doi.org/10.1093/jnci/88.3-4.153

(4) Haisma, H. J., Boven, E., van Muijen, M., de Jong, J., van der Vijgh, W. J.,

& Pinedo, H. M. (1992). A monoclonal antibody-beta-glucuronidase

conjugate as activator of the prodrug epirubicin-glucuronide for specific

treatment of cancer. British Journal of Cancer, 66(3), 474–478.

(5) Wang, S. M., Chern, J. W., Yeh, M. Y., Ng, J. C., Tung, E., & Roffler, S. R.

(1992). Specific activation of glucuronide prodrugs by antibody-

targeted enzyme conjugates for cancer therapy. Cancer Research,

52(16), 4484–4491.

(6) Mucopolysaccharidosis type VII. (2015, April 6). Retrieved April 14, 2015,

from http://ghr.nlm.nih.gov/condition/mucopolysaccharidosis-type-vii

(7) Keefe, A. D., Pai, S., & Ellington, A. (2010). Aptamers as therapeutics.

Nature Reviews Drug Discovery, 9(7), 537–550.

http://doi.org/10.1038/nrd3141

(8) Training our sights on cancer cells. (n.d.). Retrieved April 14, 2015, from

http://www.research.bayer.com/en/training-our-sights-on-cancer-cells.aspx

(9) Kaddour, S. (n.d.). Aptamer Project Site - 10% APS for Everyone: Nucleic

Acid Aptamer Selection Using Beta-glucuronidase for Monitoring Gene

Expression. Retrieved from

http://aptamerstream.blogspot.com/2013/09/nucleic-acid-aptamer-

selection-using.html